The objective of the proposed research is to describe in detail the activation and the mechanism of action of a group of metal activated kinases and related enzymes and to determine the role(s) which these cations play in the activation and the catalytic processes of these enzymes. Of the enzymes to be studied, muscle and yeast pyruvate kinase, phosphoenolpyruvate carboxykinase, enolase, and tetrahydrofolate synthetase require a divalent cation for activity and yeast pyruvate kinase and prothrombin require a divalent cation for activity and yeast pyruvate kinase and prothrombin require a divalent cation for activation. The paramagnetic cation, Mn2+ activates all of these enzymes and Gd3+ activates the prothrombin-thrombin system. In the binary enzyme-Mn2+ complex and in the ternary and higher enzyme-Mn2+-ligand complexes containing substrates, inhibitors or activators, a study of the effect of the bound paramagnetic metal on the relaxation rates of magnetic nuclei of the ligand, the solvent or the protein by nuclear magnetic resonance can yield kinetic, thermodynamic, and structural information about the environment of the cation. Electron paramagnetic resonance studies can yield thermodynamic data concerning the formation of the metal-ligand complexes and information concerning the geometry and symmetry of the metal ion when it is coordinated with the protein and protein-ligand complexes. This information will give further details as to the structure, the activation, and the mechanism of action of these enzyme systems. Enzymes which are activated by monovalent cations such as both pyruvate kinases, carboxykinase, and tetrahydrofolate synthetase, can use either T1+ or monomethylammonium ion. The NMR signals of these cations can lead to information concerning the location and environment and hence the function of these cations. These monovalent cations are a sensitive probe to obtain kinetic, structural and thermodynamic information about the monovalent cation-enzyme interaction and the location of the monovalent cation.